Pathogen detection and pathogen separation are increasingly important in the diagnosis and treatment of various diseases. Measurement and diagnostic techniques, such as flow cytometry and immunoassay panel tests, are of considerable interest in the assessment of food, water, and environmental quality.
Fluorescent and magnetic polymeric particles have known utility as markers and indicators in various biomedical assays. Among the most commonly used markers for sorting cells are immunoconjugates or immunological labels which include, for example, immunofluorescent and immunomagnetic labels. Immunofluorescent labels typically include, for example, a fluorescent molecule joined to an antibody. Immunomagnetic labels typically include, for example, a superparamagnetic particle joined to either a primary or secondary antibody. Cell labeling can be performed by, for example, attaching the antibody to a marker of interest (e.g., receptor site) on the surface of the cell, that is, a cell surface marker. However, the chemical and physical structure of cell surface marker and density of immunological labels attached to the cell surface can be difficult to accurately determine.
Fluorescent labels can be prepared, for example, by embedding or covalently coupling a fluorescent dye onto a polymeric particle. The resulting fluorescent microparticles can be analyzed manually or by other methods known in the art but preferably using an automated technique, e.g., flow cytometry, such as disclosed in U.S. Pat. No. 4,665,024, to Mansour, et al. The versatility of the fluorescent particles can be further enhanced by the incorporation of multiple fluorescent materials in a single particle. However, while simple absorption of a single dye into a particle can be adequate for most purposes, problems can arise when more than one dye is absorbed into a particle, including: inconsistent emissions attributable to, for example, intermolecular fluorescent energy transfer; differential fluorophore uptake ratios attributable to different dye solubilities within the polymeric matrix; and substrate induced changes in either or both the absorption and emission spectrum of the intercalated fluorophore.
Magnetic particles, such as known magnetically active materials, can be bonded or attached to, for example, antibodies, such as, monoclonal antibodies that are specific to a particular cell type, antigen, or other targets. The resulting magnetic-antibodies can then be mixed with a large population of many different cell types, for example, crude tissue samples, cells grown in a reactor, and the like. The magnetic-antibodies therefore attach only to their pre-selected target cell type, forming a magnetic-antibody-cell conjugate. The conjugate can then be separated from the rest of the cell population using a magnetic field. A shortcoming of magnetic particles is lack of specificity in magnetic labeling in that a cell or other biological target analyte may be rendered paramagnetic by a number of different routes which can confound the analysis and diagnostic information afforded by the method, for example, by binding a specific paramagnetic particle to a specific hapten on a cell or by specific or non-specific binding of a paramagnetic metal or metal complex directly to a cell, such as, a metal binding microorganism or by phagocytosis. Other problems encountered with magnetic particles used in detection and diagnostics include, for example, difficulty in obtaining highly accurate quantification of a cell population's magnetic susceptibility. In addition to their magnetic properties (i.e. magnetic, paramagnetic, and superparamagnetic) magnetic-antibodies can be classified, for example, into three broad categories based on their relative descending size: magnetic particulate labels, colloidal magnetic labels, and molecular magnetic labels, see for example U.S. Pat. No. 6,412,359.
There is currently a need for improved nanoparticulate materials and for methods for detection and analysis therewith, including screening methods which are highly portable for use outside the laboratory setting.